Unraveling proteoform complexity by native liquid chromatography-mass spectrometry

Proteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins can be used as medicine in health care or for their enzymatic function in the food industry. All these proteins do not exist as a single species but rather as a complex mixture of structural variants, so-called proteoforms. This heterogeneity results mainly from the presence of post-translational modifications (PTMs), such as glycosylation and glycation. To further complicate this matter, these PTMs can induce structural as well as functional changes. To allow in-depth structural and functional characterization of these proteoforms, novel analytical approaches are required to resolve proteoform heterogeneity while persevering protein nativity. The hyphenation of native separation techniques with mass spectrometry has emerged as a powerful approach to reliably study these aspects. The work in this thesis describes the (further) development and application of such methodologies for biopharmaceutical and biotechnological products.This work was supported by Dutch Research Council (NWO), SATIN project. Grant number: 731.017.202.LUMC / Geneeskund

Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry

Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batche

Anion exchange chromatography - Mass spectrometry for monitoring multiple quality attributes of erythropoietin biopharmaceuticals

Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N-ace-tyllactosamine units, was possible while providing additional information on other modifications such as O-acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs. (C) 2020 The Authors. Published by Elsevier B.V.Proteomic

Native structural and functional proteoform characterization of the prolyl-alanyl-specific endoprotease EndoPro from Aspergillus niger

Proteomic

Functional selectivity of adenosine receptor ligands

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins

Unraveling proteoform complexity by native liquid chromatography-mass spectrometry

Proteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins can be used as medicine in health care or for their enzymatic function in the food industry. All these proteins do not exist as a single species but rather as a complex mixture of structural variants, so-called proteoforms. This heterogeneity results mainly from the presence of post-translational modifications (PTMs), such as glycosylation and glycation. To further complicate this matter, these PTMs can induce structural as well as functional changes. To allow in-depth structural and functional characterization of these proteoforms, novel analytical approaches are required to resolve proteoform heterogeneity while persevering protein nativity. The hyphenation of native separation techniques with mass spectrometry has emerged as a powerful approach to reliably study these aspects. The work in this thesis describes the (further) development and application of such methodologies for biopharmaceutical and biotechnological products.</p

Unraveling proteoform complexity by native liquid chromatography-mass spectrometry

Proteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins can be used as medicine in health care or for their enzymatic function in the food industry. All these proteins do not exist as a single species but rather as a complex mixture of structural variants, so-called proteoforms. This heterogeneity results mainly from the presence of post-translational modifications (PTMs), such as glycosylation and glycation. To further complicate this matter, these PTMs can induce structural as well as functional changes. To allow in-depth structural and functional characterization of these proteoforms, novel analytical approaches are required to resolve proteoform heterogeneity while persevering protein nativity. The hyphenation of native separation techniques with mass spectrometry has emerged as a powerful approach to reliably study these aspects. The work in this thesis describes the (further) development and application of such methodologies for biopharmaceutical and biotechnological products.</p

Studying protein structure and function by native separation-mass spectrometry

Alterations in protein structure may have profound effects on biological function. Analytical techniques that permit characterization of proteins while maintaining their conformational and functional state are crucial for studying changes in the higher order structure of proteins and for establishing structure-function relationships. Coupling of native protein separations with mass spectrometry is emerging rapidly as a powerful approach to study these aspects in a reliable, fast and straightforward way. This Review presents the available native separation modes for proteins, covers practical considerations on the hyphenation of these separations with mass spectrometry and highlights the involvement of affinity-based separations to simultaneously obtain structural and functional information of proteins. The impact of these approaches is emphasized by selected applications addressing biomedical and biopharmaceutical research questions